Search

The auxiliary subunit α 2 δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α 2 δ2 mRNA is expressed in neonatal and mature hair cells. A functional α 2 δ2-null mouse, the ducky mouse (du), showed elevated auditory brainstem response click and frequency-dependent hearing thresholds. Otoacoustic emissions were not impaired pointing to normal outer hair cell function. Peak Ca 2+ and Ba 2+ currents of mature du/du inner hair cells (IHCs) were reduced by 30-40%, respectively, and gating properties, such as the voltage of half-maximum activation and voltage sensitivity, were altered, indicating that Ca v 1.3 channels normally coassemble with α 2 δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca 2+ currents. Ca 2+ - and voltage-dependent K + (BK) currents and the expression of the pore-forming BKα protein were normal. Ca v 1.3 and Ca v β2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Ca v 1.3 clusters with postsynaptic glutamate receptor GluA4 and PSD-95 clusters was significantly impaired in du/du mice. This implies that, in addition to controlling the expression and gating properties of Ca v 1.3 channels, the largely extracellularly localized α 2 δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca 2+ channels and postsynaptic AMPA receptors., Significance Statement: Inner hair cells possess calcium channels that are essential for transmitting sound information into synaptic transmitter release. Voltage-gated calcium channels can coassemble with auxiliary subunit α 2 δ isoforms 1-4. We found that hair cells of the mouse express the auxiliary subunit α 2 δ2, which is needed for normal hearing thresholds. Using a mouse model with a mutant, nonfunctional α 2 δ2 protein, we showed that the α 2 δ2 protein is necessary for normal calcium currents and exocytosis in inner hair cells. Unexpectedly, the α 2 δ2 protein is moreover required for the optimal spatial alignment of presynaptic calcium channels and postsynaptic glutamate receptor proteins across the synaptic cleft. This suggests that α 2 δ2 plays a novel role in organizing the synapse., (Copyright © 2016 the authors 0270-6474/16/3611024-13$15.00/0.)

The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (DeltaC(m)) in low-frequency (apical region, approximately 300 Hz) and high-frequency (basal, approximately 30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mm extracellular Ca(2+) and body temperature). With maturation, the Ca(2+) efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca(2+) cooperativity of exocytosis despite the smaller DeltaC(m) in apical cells. After maturation, DeltaC(m) in high-frequency IHCs increased linearly with the Ca(2+) current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca(2+) sensor otoferlin or the spatial coupling between Ca(2+) channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.

Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.

Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+- and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs.